The bond between biotin and streptavidin is one of the strongest known non-covalent interactions with a Kd of ˜10−14 M. This interaction has been developed into a streptavidin-biotin technology by functionalizing either or both of these two molecules, thereby allowing for targeting, labelling and tagging of a specific molecule. Streptavidin-biotin technology is a cornerstone of bin-assays, biomolecule purification, biomolecule immobilization, and cell separation in biotechnology and nanobiotechnology (See S. T. Kim et al., Nano Letters, 2010, 10, 2877-2833 for example). In the streptavidin-biotin technology, streptavidin is used as an anchor molecule to attach a biotinylated probe, receptor, ligand, antibody, and aptamer to a surface. (See J. Spinke et al., Langmuir, 1993, 9, 1821-1825; S. A. Walper et al., J Immunol Methods, 2013, 388, 68-77).
Streptavidin-biotin technology has also been used to form streptavidin 2D crystal substrates for visualizing biomolecular processes with Atomic Force Microscopy. (See D. Yamamoto et al., Biophys J., 2009, 97, 2358-2367). In one example of the streptavidin-biotin technology, streptavidin coated magnetic beads have been used in separation and analysis of biotinylated DNA samples. (See J. P. Ross et al., Epigenetics, 2013, 8, 113-127; M. S. Akhras et al., PLoS One, 2013, 8, e76696). Additionally, the technology has also been used as a linker to form multivalent ligands to enhance the lectin glycan interaction (see L. P. Rodriguez et al., Anal Chem, 2013, 85, 2340-2347) and in pretargeting for radioimmunotherapy (see H. B. Breitz et al., J Nucl. Med, 2000, 41, 131-140; E. Frampas, Frontiers in oncology, 2013, 3, 159.) and drug delivery (see J. Bushman et al., Journal of controlled release, 2013). Moreover, a dye labeled streptavidin may be used as a reagent for bioassays (see Z. Cao et al., Anal Chem, 2013, 85, 2340-2347) and as a marker for biotinylated cells in cell culture (see K. Tanaka et al., Bioorg Med Chem, 2012, 20, 1865-1868). In another example, the streptavidin-biotin technology has also been used in self-assembly of protein networks (See P. Ringler et al., Science, 2003, 302, 106-109) which may simplify development of new nanomaterials.
Dual biotinylated oligonucleotides were immobilized on streptavidin coated magnetic beads for PCR assays, yet some of the oligonucleotides dissociated from the beads during the thermal cycling (See D. Dressman et al., PNAS, 2003, 100, 8817-8822), Biotin dimers and trimers have been synthesized using PEG as linkers and assayed for their ability to crosslink streptavidin, and, thus, for their potential to be used to increase the amount of radioactivity on cancer cells in tumor pretargeting protocols. (See D. S. Wilbur et al., Bioconjugate Chem, 1997, 8, 819-832). A bisbiotin reagent has been used to improve efficacy of the pretargeting tumor treatment. (See K. J. Hamblett et al., Bioconjugate Chem., 2005, 16, 131-138). A ferrous complex containing two bisbiotin moieties forms a one-dimensional metal-organic framework with streptavidin as a collagen mimetic for the biomineralization of calcite. (See S. Burazerovic et al., Angew Chem Int Ed Engl, 2007, 46, 5510-5514). A heme-bisbiotin trifunctional linker has been reported as a prosthetic group capable of assembling one dimensional protein fibers. (See FIG. 2 of K. Oohara et al., Angnew Chem Int Ed Engl, 2012, 51, 3818-3821). In addition, a flexible bisbiotin peptide linker attached to alkaline phosphatase forms the ring shaped complexes with streptavidin. (see Y. Mori et al., Org Biomol Chem, 2013, 11, 914-922).
Accordingly, the potential for bisbiotin linkers is vast. However, known bisbiotin linkers lack demonstrable thermal stability. Accordingly, there remains a need in the art for thermostable bisbiotin linkers.